Chromatography Apparatus And Methods Using Multiple Microfluidic Substrates

ABSTRACT

An apparatus for chemical separations includes a first substantially rigid microfluidic substrate defining a first fluidic port; a second substantially rigid microfluidic substrate defining a second fluidic port; and a coupler disposed between the first and second substrates, the coupler defining a fluidic path in fluidic alignment with the ports of the first and second substrates. The coupler includes a material that is deformable relative to a material of the first substrate and a material of the second substrate. The substrates are clamped together to compress the coupler between the substrates and form a fluid-tight seal.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.Nos. 61/182,268 and 61/182,498, both filed on May 29, 2009, and is acontinuation-in-part of PCT International Application No.PCT/US10/26352, filed on Mar. 5, 2010 and designating the U.S. Theentire contents of these applications are incorporated herein byreference.

FIELD OF THE INVENTION

The invention relates generally to chromatography. More specifically,the invention relates to liquid-chromatography utilizing multiplemicrofluidic substrates.

BACKGROUND

High-performance liquid chromatography (HPLC) instruments are analyticaltools for separating, identifying, and quantifying compounds.Traditional HPLC instruments use analytical columns constructed fromstainless-steel tubing. Typically, the tubing has an inner bore diameterof 4.7 mm, and its length ranges from about 5 cm to about 25 cm.

In addition, the analytical column of an HPLC instrument typically has afritted end fitting attached to a piece of tubing. Particles, typicallysilica-based, functionalized with a variety of functional moieties, packthe tube.

To achieve optimal separation efficiency, using the completed column, anappropriate flow rate of a mobile phase is important. For a 4.7 mmdiameter column packed with 5 μm diameter particles, a desirable flowrate is typically between about 1 mL/min and about 2 mL/min. Minimizingthe presence of unswept dead volume in the plumbing of the HPLCinstrument is desirable for maintaining separation efficiency.

In an HPLC instrument, an injector is typically used to inject a sampleinto a flowing mobile phase as a discrete fluidic plug. Dispersion of aplug band as it travels to and/or from the column reduces the ultimateefficiency of the chromatographic system. For example, in achromatographic system using 4.7 mm column tubing and a mobile phaseflowing at 1-2 mL/min, tubing having an outer diameter of 1/16 inch andan inner diameter of about 0.010 inch is typically used to plumbconnections between the various HPLC components (e.g. pump, injector,column, and detector). For these flow rates and tubing dimensions, it isrelatively easy to machine port details to tolerances that will ensureminimal band broadening at tubing interfaces.

A desire to reduce mobile-phase solvent consumption, in part, hasmotivated a trend towards reducing column inner diameter. Thus, severalscales of chromatography are now commonly practiced; these are typicallydefined as shown in Table 1 (where ID is inner diameter.)

TABLE 1 HPLC Scale Column ID Typical Flow range Analytical 4.7 mm 1smL/min Microbore 1-2 mm 100s μL/min Capillary 300-500 μm 10s μL/min Nano50-150 μm 100s nL/min

Microbore HPLC has often been practiced with equipment similar to thatused for analytical scale HPLC, with minor modifications. Aside fromrequiring the exercise of a small degree of additional care in makingfittings, microbore HPLC typically requires an operating skill levelsimilar to that of analytical scale HPLC.

In contrast, capillary and nano-scale HPLC require relativelysignificant changes in HPLC components relative to analytical-scaleHPLC. Generation of stable mobile-phase flows of less than about 50μL/min is relatively difficult using standard open-loop reciprocatingHPLC pumps, such as those commonly found in analytical and microboreHPLC systems.

For capillary-scale chromatography, stainless-steel tubing is usable forcomponent interconnections; however, the inner diameter must typicallybe less than 0.005 inch (less than about 125 μm). Care is generallyrequired in the manufacture of fitting terminations to avoid creation ofeven minute amounts of dead volume.

For nano-scale chromatography, tubing having inner diameters of about25-50 μm is typically required to interconnect components of aninstrument (e.g., to connect a pump to a separation column). Becausestainless-steel tubing is typically unavailable in these dimensions,polyimide-coated fused-silica tubing is typically used. Althoughfused-silica tubing has excellent dimensional tolerances and very clean,non-reactive interior walls, it is fragile and can be difficult to workwith. In addition, interconnection ports should be machined to exactingtolerances to prevent even nanoliters of unswept dead volume.

While the primary motivation to replace analytical-scale HPLC withmicrobore-scale HPLC may be the desire for reduced solvent consumption,moving to capillary-scale and nano-scale chromatography can supportimproved detection sensitivity for mass spectrometers, in addition tofurther reducing solvent consumption, when, for example, flows of lessthan about 10 μL/min are used. Moreover, capillary-scale or nano-scalesystems are often the only options for the sensitive detection typicallyrequired for applications involving small amounts of available sample(e.g., neonatal blood screening).

Despite the advantages of capillary-scale and nano-scale chromatography,HPLC users tend to employ microbore-scale and analytical-scalechromatography systems. As described above, these systems typicallyprovide good reliability and relative ease-of-use. In contrast,maintenance of good chromatographic efficiency while operating acapillary-scale or nano-scale chromatographic system requiressignificant care when plumbing the system (e.g., using tubing to connectpump, injector, column, and detector).

In practice, an operator switching from an analytical or microbore-scalesystem to a capillary or nano-scale system at times finds that betterseparation efficiency was achieved with the higher-flow rate (i.e., theanalytical or microbore-scale) system. This typically occurs due toinsufficiency in the operator's knowledge or experience required toachieve low band-spreading tubing interconnections. Moreover, use ofsmaller inner-diameter tubing at times can lead to frequent plugging oftubing.

Due the relative difficulty typically encountered with capillary-scaleHPLC systems and, even more so, with nano-scale HPLC systems, suchsystems have primarily been used only when necessary, such as for smallsample sizes, and when a relatively skilled operator is available. Thus,analytical laboratories tend to possess more analytical-scale andmicrobore-scale systems than capillary-scale and nano-scale systems, anddo not realize the full benefits available from capillary-scale andnano-scale HPLC.

Proteomic analyses often utilize a trap column for sample enrichment andcleaning prior to separation of the sample in an analytical column.Often, different packing material chemistries are used for the trap andseparation columns; sample components trapped on the trap column may beserially driven from the trap to the separation column during agradient-based mobile phase elution process. The components can beinitially focused at the head of the analytical column, due to thedifferent chemistry, until the gradient attains a level that drives thecomponent from the chemistry of the analytical column. It is also commonto place the analytical column in an oven to provide a stable, elevatedtemperature, which promotes elution of sample components from theanalytical column.

Some chromatography instruments utilize a microfluidic substrate. Suchsubstrates can ease handling of small samples and reduce undesirableeffects such as dispersion.

SUMMARY

Some embodiments of the invention arise, in part, from the realizationthat a microfluidic analytical apparatus can advantageously employee twoor more microfluidic substrates. Multiple substrates can be used, forexample, for thermal isolation and/or pre-loading of samples. Multiplesubstrates can implement, for example, a trap on one substrate, and ananalytical column on another substrate, or can implement an infusioncolumn on one substrate and a calibration column on a second substrate.

Some embodiments of the invention arise, in part, from the realizationthat two substrates can be fluidically coupled via mechanical contactswith a coupling component, formed, for example, from a polymer material.

Some embodiments arise, in part, from a realization that an integratedhigh-pressure chemical-separation device, such as an HPLC instrument, isadvantageously fabricated, in part, from sintered inorganic particles.Some embodiments of the invention provide nano-scale microfluidic LCinstruments that offer integration of a trap/enrichment column(s) and aseparation column(s) on one (or more) ceramic-based substrates; some ofthese embodiments include features that support cooling or thermalisolation of the enrichment column to enhance cycle time and/or improveenrichment-column performance.

Various embodiments of the invention provide one or more manufacturingor functionality advantages. Potential manufacturing advantages includereduction in substrate complexity, simplification of packing processes,size reductions, use of mixed materials (for example, differentmaterials in the different substrates, and increased apparatus yield.)Potential functionality advantages include thermal decoupling ofdifferent heating/cooling zones, a slot design for loading a secondsubstrate, reduced clamping force, a modular design with replaceable orreconfigurable components, and increased design flexibility (forexample, for multiple traps and an internal sample loop.)

Accordingly, in one aspect, the invention features an apparatus forchemical separations. The apparatus includes a first substantially rigidmicrofluidic substrate defining a first fluidic port; a secondsubstantially rigid microfluidic substrate defining a second fluidicport; and a coupler disposed between the first and second substrates,the coupler defining a fluidic path in fluidic alignment with the portsof the first and second substrates. The coupler includes a material thatis deformable relative to a material of the first substrate and amaterial of the second substrate.

Other aspects of the invention relate to methods of making amicrofluidic-based apparatus and methods of microfluidic-based analysis.Some preferred embodiments include a mass analyzer.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and further advantages of this invention may be betterunderstood by referring to the following description in conjunction withthe accompanying drawings, in which like numerals indicate likestructural elements and features in various figures. The drawings arenot necessarily to scale, emphasis instead being placed uponillustrating the principles of the invention.

FIG. 1 is a front view of an embodiment of a liquid chromatography-massspectrometer system including a liquid chromatography module with aninstalled microfluidic cartridge.

FIG. 2 is a front view of an embodiment of the liquid chromatographymodule of FIG. 1.

FIG. 3 is a view of the liquid chromatography module of FIG. 2 with anopen cover to show a clamping assembly housed within.

FIG. 4 is an isometric view of an embodiment of the clamping assembly ofFIG. 3 housed within the liquid chromatography module.

FIG. 5 is a side view of the clamping assembly of FIG. 3.

FIG. 6 is a front view of the clamping assembly of FIG. 3.

FIG. 7 is a view of an embodiment of an end housing of the clampingassembly of FIG. 3.

FIG. 8 is an exploded isometric view of the end housing of FIG. 7.

FIG. 9 is an exterior view of an alternative embodiment of a back wallfor an end housing.

FIG. 10 is an interior view of the alternative embodiment of a back wallfor the end housing of FIG. 9.

FIG. 11 is a view of the right side of one embodiment of a microfluidiccartridge.

FIG. 12 is a view of the left side of the microfluidic cartridge of FIG.11.

FIG. 13 is an exploded view of the microfluidic cartridge of FIG. 11.

FIG. 14 is a side view of the microfluidic cartridge of FIG. 11 with theright side removed.

FIG. 15 is another side view of the microfluidic cartridge of FIG. 11with the right side removed, showing a push block superimposed upon themicrofluidic substrate in the microfluidic cartridge.

FIG. 16 is a side view of a variant of the microfluidic cartridge ofFIG. 11 with the left side removed.

FIG. 17 is a side view of one embodiment of a microfluidic substratewithin the microfluidic cartridge of FIG. 11.

FIG. 18A is a view of an embodiment of a separation device having morethan one microfluidic substrate.

FIG. 18B is a cross-sectional view of a portion of the device of FIG.18A, illustrating, in more detail, fluidic-connection and alignmentfeatures.

FIG. 19 is a view of an embodiment of an infusion device having morethan one microfluidic substrate.

FIG. 20 is a cross-sectional front view of the clamping assembly withoutan installed microfluidic cartridge.

FIG. 21 is a cross-sectional front view of the clamping assembly with amicrofluidic cartridge installed therein, and with the clamping assemblyin an unclamped position.

FIG. 22 is a cross-sectional front view of the clamping assembly with amicrofluidic cartridge inserted therein, and with the clamping assemblyin a clamped position.

FIG. 23 is a view of the emitter end of the microfluidic cartridge, witha high-voltage cable and a gas nozzle coupled to a side thereof.

DETAILED DESCRIPTION

Preferred high-performance liquid chromatography (HPLC) andultra-high-pressure LC (UHPLC) apparatus, some non-limiting examples ofwhich are described herein, have an installation chamber for receiving amicrofluidic cartridge; the cartridge optionally has an electrosprayemitter, and chamber postions the tip of the emitter into operablecommunication with mass-spectroscopy components of the apparatus. Themicrofluidic cartridge houses two or more substantially rigidmicrofluidic substrates. For protein samples, the ceramic is preferablya High-Temperature Co-fired Ceramic (HTCC), which provides suitably lowlevels of loss of sample due to attachment of sample to walls ofconduits in the substrate.

A channel, formed in the layers of the substrate, operates as aseparation column. Apertures in the side of the substrate—formed, forexample, via laser etching—provide openings into the channel throughwhich fluid may be introduced into the column. Fluid passes through theapertures under high pressure and flows toward the electrospray emittercoupled at the egress end of the channel. Holes in the side of themicrofluidic cartridge provide fluidic inlet ports for delivering fluidto the substrate. Each of one or more fluidic inlet ports align with andencircle one of the fluidic apertures.

A clamping mechanism applies a mechanical force to one side of themicrofluidic cartridge, urging the substrate against fluidic nozzlescoupled to the installation chamber. The nozzles deliver fluid to thesubstrate through the fluidic inlet ports of the cartridge.

Various embodiments, arise, in part, from a realization that variouscomponents, such as ports, nozzles and connectors are desirably formedof a deformable material, such as a polymer, and that a mechanical forceof sufficient strength can be applied to a substantially rigid substrateto produce a tight, non-leaking seal between each nozzle and the surfaceof the substrate encircling an aperture. Preferably, the appliedpressure, at the contact surface between the substrate and a tube, isgreater than the pressure of a fluid passing through the tube into thesubstrate. A suitable polymer is, for example, polyether-ether-ketone,such as PEEK™ polymer (available from Victrex PLC, Lancashire, UnitedKingdom.)

Because a ceramic-based substrate may be prone to fracture if subjectedto a mechanical force focused at a single small point and/or applied ina manner that tends to introduce shear stress (such as by tending tobend and/or twist the substrate,) the clamping mechanism preferablyemploys a multi-surfaced probe and/or preferably counters a forceapplied to (and perpendicular to) one side of the substrate with anequal, substantially collinear force applied to the opposite side of thesubstrate, in a manner to introduce compressive stress substantiallywithout shear stress.

A multi-surfaced probe, for example, presses against the substrate atmultiple points of contact simultaneously. Thus, a probe is preferablyconfigured to contact the substrate in a manner that tends to distributeforces and reduce or eliminate the potential for shear stress.Preferably, multiple contact sites associated with a probe are alignedwith features that contact the opposite side of the substrate, to thusmitigate or eliminate introduction of shear stress by the clampingmechanism.

Any or all of the features that contact the substrate, from either side,optionally include conduits, for gases and/or liquids, and optionallyinclude electrical conductors, and/or optical conductors, and/or othercommunication pathways.

The multiple points of simultaneous contact optionally distribute themechanical force over a greater area than that of a single point ofcontact. Preferably, the points of contact are associated withsubstantially equidistant points on a circle, and/or define a circularpattern of force distribution. Preferably, a component that contacts asubstrate at multiple points receives an applied force at a single site,thus potentially reducing the likelihood or degree of twisting forcesapplied to a substrate. Further, the substrate preferably has somefreedom of movement within the microfluidic cartridge, being free tofloat until the clamping mechanism is engaged, thus permitting thesubstrate to “self-adjust” its position during the clamping process sothat stresses, other than compressive, do not impinge upon thesubstrate, and a housing portion of the cartridge does not applysubstantial, if any, force to the substrate.

In addition to the substrate, the microfluidic cartridge houses internalcircuitry and a temperature control unit for heating and cooling thesubstrate. An aperture in the microfluidic cartridge provides a windowthrough which pogo pins supply low voltage and other electrical signalsto internal circuitry. Another aperture in the microfluidic cartridge,near the tip of the electrospray emitter, operates as a gas inlet portthat couples to a gas nozzle. Still another aperture, disposed near theemitter tip, serves as a high-voltage input port. A high-voltage cablecouples to this high-voltage input port to deliver high voltage to thetip region of the emitter, for example, a voltage of approximately 3keV.

The mechanical force used to urge the tubing against the substrate alsooperates to establish connections between the high-voltage cable and thehigh-voltage input port, between the electrically conductive pogo pinsand an electrical connector, and between the gas nozzle and the gasinlet port. Thus, a single act of clamping the microfluidic cartridgewithin the installation chamber concurrently establishes the variousfluidic and electrical connections needed for operating the separationcolumn.

FIG. 1 shows a front view of one embodiment of a liquidchromatography-mass spectroscopy (LC-MS) system 10 in which theinvention may be embodied. The LC-MS system 10 includes a liquidchromatography module 12 having a slot 14 within which resides a fullyinstalled microfluidic cartridge 16. As shown, the handle of themicrofluidic cartridge 16 projects from the slot 14. The liquidchromatography module 12 is coupled to a mass spectroscopy (MS) unit 18.In one embodiment, the LC-MS system 10 is a modified version of ananoACQUITY UPLC® system produced by Waters Corporation of Milford,Mass.

FIG. 2 shows a front view of an embodiment of the liquid chromatographymodule 12 having a housing 20. The housing 20 has an upper section 22with an on-off switch 23 and with status and pressure indicators 24, amiddle section 26 having the slot 14 for receiving the microfluidiccartridge and an arm portion 28, and a lower section 30 having anadjustment knob 32 extending from an opening in the housing. Theadjustment knob 32 is coupled to an interior x-translation stage (partlyvisible) for moving the microfluidic cartridge 16 along the x-axis.Adjustment knobs for y-translation and z-translation stages (not shown)also enable y-axis and z-axis adjustments of the position of themicrofluidic cartridge relative to the MS unit 18 (FIG. 1). Suchadjustments provide, for example, positioning of an emitter tip outletin relation to an MS inlet orifice.

Coupled to the arm portion 28 is a lever 34 that is rotatable about apivot point 36 between a clamped position and an unclamped position. InFIG. 2, the lever is in the unclamped position. Counterclockwiserotation of the lever about the pivot point, approximately 180 degrees,moves the lever into the clamped position. At one end of the housing, anelectrical cable 38 and an electrical signal conductor 40 enter thehousing through an opening 42 in the front of the housing. Theelectrical cable 38 supplies a high voltage, and the electrical signalconductor 40 supplies a low voltage, to the microfluidic cartridge, asdescribed herein. Not shown are the microfluidic tubing and a gas line,which also enter the housing through the opening 42, for bringing fluidand gas, respectively, to the microfluidic cartridge.

FIG. 3 shows the housing 20 with its front cover 50 opened to expose aclamping assembly 60 within. A hinge along a base edge of the housingattaches the front cover 50 to a translation stage support 52. Thetranslation stages and adjustment knobs are absent from the drawing tosimplify the illustration. Other components residing within the housinginclude a circuit board 62. The translation stage support 52, clampingassembly 60, and circuit board 62 are coupled to a rear panel 64 of thehousing.

The electrical cable 38 and an electrical conduit 66 couple to one sideof the clamping assembly 60. The electrical cable carries a high voltage(e.g., 3000 volts), and the electrical conduit 66 bundles a plurality oflow-voltage electrical conductors. Not shown are the microfluidic tubingand gas line that are also coupled to the same side of the clampingassembly 60 as the electrical cable 38 and electrical conduit 66.

The clamping assembly 60 has a slot 68 for receiving a microfluidiccartridge and a post 70 to which the lever 34 (FIG. 2) is attached. Whenthe front cover 50 is closed, the slot 14 in the front cover 50 alignswith the slot 68 of the clamping assembly 60, the adjustment knob 32 ofFIG. 2 (not shown in FIG. 3) projects through the opening 72 in thefront cover 50, and the post 70 projects through another opening, whichis obscured by the sidewall of the arm portion 28.

FIG. 4 shows an embodiment of the clamping assembly 60 having a body 80coupled to an end housing 82. The post 70 joins the lever 34 to one side(called herein the front side) of the body 80. The end housing 82 hasopposing sidewalls 84-1, 84-2 (generally, 84) spatially separated by aback wall 86. The sidewall 84-2 is not visible; the reference numeralpoints generally to the area of the side wall 84-2. Sidewall 84-1 hasthe slot 68 (FIG. 3) that is adapted to receive the microfluidiccartridge 16 (FIG. 1). Sidewall 84-2 has a corresponding slot (notshown) aligned with the slot 68 in the sidewall 84-1 such that themicrofluidic cartridge 16 passes through both slots upon being installedinto the clamping assembly 60.

The back wall 86 of the end housing 82 has a pogo pin block 88 and afluidic block 90. The pogo pin block 88 includes a two-piece bracket 92,joined by fasteners 94, for retaining the electrical conduit 66 (notshown) therebetween. The pogo pin block 88, mostly obscured in FIG. 4 bythe two-piece bracket 92, is disposed adjacent to and above the fluidicblock 90. This embodiment of fluidic block 90 has three apertures 96 forreceiving the ends of tubes that deliver fluid. A spacer block 98secures the pogo pin block 88 and fluidic block 90 within a slot (shownin FIG. 8) in the back wall.

Projecting from a surface of the back wall 86 is an L-shaped retainer100 having a major surface 102 with three openings 104, 106, 108therein. The opening 104 is for retaining a gas line (not shown) that iscoupled to the clamping assembly 60; the opening 106 is for retainingthe high-voltage electrical cable 38 (FIG. 3), and the opening 108 isfor receiving a fastener that joins the retainer 100 to the back wall86. Extending from the rear side of the clamping assembly 60 (i.e., theside presented to the MS unit 18 of FIG. 1) is an arm 110 used torestrict the extent to which the microfluidic cartridge 16 can beinserted through the slots 68.

FIG. 5 shows a side view of the clamping assembly 60, with themicrofluidic cartridge 16 passing through both slots in the sidewalls 84of the clamping assembly 60. The arm 110 catches a nook in an upper edgeof the microfluidic cartridge 16, preventing the microfluidic cartridgefrom sliding further through the slots 68. The high-voltage electricalcable 38 couples to the opening 106 and the electrical conduit 66couples to the two-piece bracket 92 of the pogo-pin block. In addition,a coupler 112 connects to the opening 104 in the L-shaped retainer 100.

FIG. 6 shows a front view of the clamping assembly 60 and a chamber 120visible through the slots 68. Within the chamber 120 is a carriage 122for receiving the microfluidic cartridge 16. Extending inwardly into thechamber 120 from one side (i.e., in FIG. 6, the right side) of thecarriage 122 are upper and lower springs 124-1, 124-2, respectively, andthe tip of a plunger 126. Extending inwardly into the chamber 120 fromthe opposite side (i.e., from the direction of the back wall 86 of theend housing 82) is a guide pin 128 and a plurality of microfluidicnozzle tips 130-1, 130-2. In this embodiment, the microfluidic nozzletip 130-2 obscures a third microfluidic nozzle tip 130-3 (FIG. 7), whichis horizontally in line with the microfluidic nozzle tip 130-2.

FIG. 7 shows an interior view of one embodiment of the end housing 82,including the opposing sidewalls 84-1, 84-2, separated by the back wall86. The sidewall 84-1 has the slot 68-1, and the sidewall 84-2 has theslot 68-2. Installed in the back wall 86 are the pogo pin block 88 andfluidic block 90.

The interior side of the pogo pin block 88 has a recessed region 140with a pogo pin electrical connector 142 projecting inwardly from asurface thereof. In this example, the electrical connector 142 has tenelectrically conductive pogo pins 144 for conducting electrical signals.Each pogo pin 144 is an individual cylindrical, spring-loaded electricalconductor for transmitting electrical signals.

The interior side of the fluidic block 90 has the plurality ofmicrofluidic nozzles 130-1, 130-2, 130-3 (generally, 130) of FIG. 6projecting therefrom. In one embodiment, the nozzles 130 are three innumber and arranged in a triangular pattern. The locations of thesenozzles 130 are fixed with respect to each other. Fluid delivered bymicrofluidic tubes to the apertures 96 (FIG. 4) on the exterior side ofthe fluidic block 90 exits through these nozzles 130. Situated below thetriangular pattern of nozzles 130, aligned with the nozzle at the apexof the triangle, is the guide pin 128.

FIG. 8 shows an exploded view of the end housing 82, to illustrate anassembly process of the back wall 86. The back wall 86 has a slot 150into which slide, in succession, the fluidic block 90, pogo pin block88, and spacer 98. The slot 150 has a lower rectangular region 152 and atiered upper region 154. The lower region 152 is adapted to receive thefluidic block 90 and pogo pin block 88. The shape of the upper region154 is adapted to receive the spacer 98. The spacer 98 has shoulders 155with holes 156 therein, through which fasteners can join the spacer torespective holes 157 in the back wall 86, thus securing the blocks 88,90, 98 within the slot 150.

FIG. 9 and FIG. 10 show an alternative embodiment of a back wall 86′ forthe end housing 82; FIG. 9 shows an exterior side of the back wall 86′,and FIG. 10 shows an interior side. In this embodiment, the back wall86′ has four nozzles to contact a microfluidic substrate rather than thethree nozzles 130 of the fluidic block 190 described in FIG. 7. The backwall 86′ includes four fluidic inlet ports 170-1, 170-2, 170-3, 170-4(generally, 170) arranged in a diamond pattern. Each fluidic inlet port170 includes a round fitting (most visible in port 170-2 of the fourports) that projects generally orthogonal from the back wall 86′. Eachfluidic inlet port 170 is floating with respect to the other ports 170;that is, the fluidic inlet ports 170 themselves are not fixed to theback wall 86′ so that each fluidic inlet port 170 can move slightly andindependently of the other ports 170.

As examples of fluidic plumbing, the tip of a microfluidic tube 172 ispress fit into fluidic inlet ports 170-1 and 170-3, whereas fluidicinlet port 170-4 is blocked with a plug 174 (i.e., unused), and fluidicinlet port 170-2 is open. The back wall 86′ also includes an alternativeembodiment of a pogo pin block 88′ having a single row of electricalconnectors 176 (here, e.g., ten in number).

FIG. 10 shows an interior side of the alternative embodiment of the backwall 86′ described in FIG. 9. The interior side of the pogo pin block88′ has a recessed region 180 from which project the row of electricallyconductive pogo pins 176. Below the row of pogo pins is a secondrecessed region 182. Projecting from this recessed region 182 are fourmicrofluidic nozzles 184, each corresponding to one of the ports 170 onthe exterior side of the back wall 86′. Fluid delivered by microfluidictubes 172 to the ports 170 on the exterior side of the back wall 86′exits through these nozzles 184. Situated below the diamond pattern ofnozzles 184 is an alignment pin 186, similar in function to the guidepin 128.

FIG. 11 shows a right side view an embodiment of the microfluidiccartridge 16 that works in conjunction with the type of nozzlearrangement described in FIG. 7. As described further below, themicrofluidic cartridge 16 houses an emitter, a microfluidic substrate, aheater, and circuitry, and operates as an electromechanical interfacefor the delivery of voltages, electrical signals, and fluids (gas andliquid) to the various components housed within the microfluidiccartridge 16.

This embodiment of microfluidic cartridge 16 is made by joining twocasing sections 200-1, 200-2, for example, by snapping the halvestogether, or using glue or mechanical fasteners, or any combinationthereof. The two casing sections are also referred to herein as the leftand right sides of the microfluidic cartridge 16, with the terms leftand right being determined by the orientation of the microfluidiccartridge 16 when it is inserted into the clamping assembly 60. It is tobe understood that such terms as left, right, top, bottom, front, andrear are for purposes of simplifying the description of the microfluidiccartridge, and not to impose any limitation on the structure of themicrofluidic cartridge itself.

The right casing section 200-1 has a grip end 202 and an emitter end204. A curved region 206 within the grip end 202 provides a finger holdby which a user can grasp the microfluidic cartridge 16 when insertingand removing it from the liquid chromatography module 12.

In the side of the casing section 200-1 is a rectangular-shaped window208, within which resides a push block 210. The surface of the pushblock 210 lies flush with the surface of the right casing section 200-1.As described further below, the push block 210 is not rigidly affixed tothe right casing section 200-1, and can move slightly in, out, up, down,left, or right; that is, the push block 210 floats within the window208. In one embodiment, the push block 210 is made of metal.

Disposed below the push block 210 is an opening 212, which extendscompletely through both casing sections 200-1, 200-2. Hereafter, theopening 212 is referred to as a through-hole 212. At the emitter end 204is a nook 214 in the top edge of the microfluidic cartridge 16. Withinthe nook 214, a movable fin 216 projects through the top edge betweenthe casing sections 200-1, 200-2.

FIG. 12 shows the left casing section 200-2 of the microfluidiccartridge 16. Like the right casing section 200-1, the left casingsection 200-2 has a grip end 202 with a curved region 206 and an emitterend 204. Approximately central in the length of the left casing section200-2 are three nozzle openings 220 in a triangular pattern that matchesthe triangular arrangement of the nozzles 130 of the fluidic block 90(FIG. 7). The triangular pattern is desirable, to define a circular loadpattern. The center of the circle is preferably aligned to the axis ofmotion of a plunger 126 (FIG. 20) of the clamping assembly 60 (FIG. 6)to provide a load balance on the metal-plate bosses 260 of the pushblock 210. A third fluidic port is used, for example, for a calibrationport; an operator can run a calibration fluid. The other two portsprovide access to, for example, the analytical column and the trapcolumn.

Concentrically located behind each nozzle opening 220 is a microscopicfluidic aperture in the side of a microfluidic substrate housed withinthe microfluidic cartridge. The fluidic conduits of the microfluidicnozzles 130 of the fluidic block 90 have much larger inner diametersthan the size of the microscopic apertures in the substrate, whichfacilitates alignment therebetween. In one embodiment, each microscopicfluidic aperture has a 0.003″ square cross section, and eachmicrofluidic nozzle 130 has a 0.013″ orifice (lumen with a circularcross section) that aligns with and circumscribes the microscopicfluidic aperture on the substrate, such as a 0.003″ via (aperture with asquare cross section.)

The microfluidic nozzles 130 utilize a polymer-to-ceramic interface,relying only on the compressive stress provided by the clamping assembly60 (FIG. 6) to provide a fluidic seal; that is, the clamping assembly 60provides a greater pressure at the polymer-to-ceramic interface than theoperating fluidic pressure. For example, for an operating fluidicpressure of 5,000 psi—or alternative pressures, such as 15,000 psi—areimplemented with a clamping load of 130 pounds across the total surfacearea of the nozzle-to-substrate interface, producing an effectivefluidic seal for the selected operating pressure.

Directly above the apex of the triangularly arranged nozzle openings 220is a rectangular depression 222 within the left casing section 200-2.The depressed region 222 surrounds a rectangular-shaped window 224through which an array of electrical contacts 226 is accessed. Theelectrical contacts 226 are electrically conductive pads for makingelectrical contact with the pogo pins 144 of the pogo pin block 88 (FIG.7). The array of electrical contacts 226 is part of a flex circuitoverlaid upon the microfluidic substrate, as described further below inconnection with FIG. 13.

At the emitter end 204, the left casing section 200-2 has a gas inletport 225 for receiving a gas nozzle and a high-voltage input port 228for receiving the tip (pogo-pin) of the high-voltage electrical cable 38(FIG. 5). A plurality of holes 234 hold alignment pins 236 that are usedto align the casing sections 200-1, 200-2 when the halves are beingjoined.

The left casing section 200-2 further includes a rectangular-shapedgroove 230 along its bottom edge. The groove 230 has an open end 232 atthe emitter end 204, extends laterally therefrom, and terminates at thethrough-hole 212 situated below the nozzle openings 220. In addition,the groove 230 receives the guide pin 128 (FIG. 7) when the microfluidiccartridge 16 is inserted into the slot 68 (FIG. 4) of the clampingassembly 60. When the guide pin 128 reaches the through-hole 212, thenthe microfluidic cartridge 16 is fully installed in the chamber 120 andin position for clamping.

FIG. 13 shows an exploded view of the microfluidic cartridge 16, and thevarious components housed within. Disposed between the right casingsection 200-1 and the left casing section 200-2 are a microfluidicsubstrate 250, an emitter assembly 252 (also referred to herein as a“spray unit”) that couples to the microfluidic substrate, and a shutter254. On the interior side of the right casing section 200-1 is arectangular recess 256 adapted to closely receive the push block 210(i.e., the push block 210 snaps into and floats within the recess 256).With the push block 210 sitting within this recess 256, a raised portionof the push block (on its opposite unseen side) enters the window 208 inthe side of the right casing section 200-1.

In addition, this embodiment of push block 210 has three raised bosses260, each with a planar face. The planar faces of the three bosses presssimultaneously against the side of the microfluidic substrate when anurging force is applied to the push block 210 from an exterior side ofthe first casing section 200-1, spreading out the force to avoid asingle concentrated point of contact. Each raised boss 260 alignsdirectly opposite one of fluidic apertures in the microfluidic substrate250, and thereby applies pressure (when the push block is pushed)directly opposite one of the nozzle openings 220 in the left casingsection 200-2, thus avoiding production of shear stresses by, forexample, twisting and or bending the microfluidic substrate 250.

Other embodiments can have more, or fewer, than three bosses. Ingeneral, the number of bosses corresponds to the number of fluidicapertures (which may include dummy apertures) in the microfluidicsubstrate 250, so that there is one boss for each fluidic aperture,aligned directly opposite that fluidic aperture. In general, the numberof bosses corresponds to the number of fluidic nozzles and dummy nozzlesthat contact the substrate 250, so that all bosses align with acorresponding nozzle. The number and arrangement of bosses and nozzlesare optionally selected to control application of undesirable stressesto the microfluidic substrate 250.

The assembly 252 includes an emitter 266, an emitter retainer 241A thatpositions and/or aligns the emitter 266 with the substrate 250, and asheath-gas component 279. The component 279 receives a sheath gas via atube 278, which is disposed in the housing sections 200-1, 200-2. Theretainer 241A aligns a lumen of the emitter 266 with an outlet port ofthe substrate 250. Preferably, additional component(s) urge the emitter266 into contact with the substrate 205, with sufficient force toprovide a greater interfacial pressure than a pressure of an eluentflowing through the outlet port into the lumen of the emitter 266.

Folded over a top edge of the microfluidic substrate 250, a flex-circuitassembly 258 includes the array of electrical contacts 226. As describedin FIG. 12, these electrical contacts 226 are accessible through thewindow 224 in the left casing section 200-2. Further, the shutter 254has holes 262 that align with the nozzle openings 220 in the side of theleft casing section 200-2 so that the microscopic fluidic apertures inthe surface of the microfluidic substrate 250 are exposed. In addition,the shutter 254 has the fin 216 (FIG. 11) and an emitter tube 264 thatpartially envelopes the electrospray emitter 266.

The substrate 250 is optionally formed in the following manner. Fivegreen-sheet layers, for multiple substrates 250, are pressed together,after desired patterning. Vias for fluidic apertures are laser etched inone or both sides of the pressed sandwich. Edge portions are defined bylaser etching. After firing, individual substrates 250 are snappedapart. Edges, or portions of edges, are optionally polished.

FIG. 14 shows a right side view of the microfluidic cartridge 16 withthe right casing section removed to reveal various components housedwithin and to show various features of the interior side of the leftcasing section 200-2. The various components include the microfluidicsubstrate 250 and the shutter 254, both of which are coupled to the leftcasing section 200-2 as shown.

On the surface of the microfluidic substrate 250 is the flex-circuitassembly 258, comprised of a control circuitry portion 257 and a heaterportion (hereafter, heater 270). The flex-circuit assembly 258 foldsover a top edge of the microfluidic substrate 250 and covers a portionof the opposite side of the microfluidic substrate 250. An integratedcircuit (IC) device 272 is mounted on the control circuitry portion ofthe flex-circuit assembly 258. In one embodiment, the IC device 272 is amemory device (e.g., EPROM) for storing program code and data. Theheater 270 covers a separation column within the microfluidic substrate250. Mounted to the heater 270 is a temperature sensor 274.

The flex-circuit assembly 258 is constructed of multiple stacked layers(e.g., three, four, or five). The polymer substrate of each layer holdsdifferent interconnectivity or circuitry. One of the layers containsresistive traces of the heater 270. Electrical contacts at the two endsof the resistive traces connect to two pads 259 on the control circuitryportion 257. Another layer of the flex-circuit assembly 258 has viasthat electrically contact the ends of the resistive traces, anotherlayer has contacts to connect electrically to electrical components 272,274, and still another layer has the pogo-pin contact pads 226 (FIG.13). Through the flex-circuit assembly 258, each of the electricalcomponents 272, 274 and resistive traces are electrically coupled to thecontact pads 226. The gas inlet port 225 opens into a well 276 thatchannels injected gas into a gas tube 278 that delivers the gas to theemitter end 204.

FIG. 15 shows the right side view of the microfluidic cartridge 16 ofFIG. 14, again with the right casing section removed, and with theadditional feature of the push block 210 (FIG. 13) suspended at theapproximate location where the push block 210 abuts the microfluidicsubstrate 250. The location is near the southwest quadrant of themicrofluidic substrate 250, directly below the flex-circuit assembly 258and behind the heater 270 (with respect to the emitter end 204 being thefront of the microfluidic cartridge 16). The push block 210 comprises asmaller rectangular block 282 disposed upon, or an integral extensionof, a larger rectangular block 280. The smaller rectangular block 282 issized to closely fit within the window 208 (FIG. 13) of the right casingsection 200-1.

FIG. 16 shows a left side view of the microfluidic cartridge 16 with theleft casing section removed to reveal various components housed withinand to show features of the interior side of the right casing section200-1. The flex-circuit assembly 258 wraps around onto this side of themicrofluidic substrate 250, and includes the array of electricalcontacts 226 of FIG. 12. In addition, the microfluidic substrate 250 hasa high-voltage input port 290 for receiving the electrically conductiveterminal of the high-voltage electrical cable 38 (FIG. 5). Thehigh-voltage input port 290 is disposed near an egress end of aseparation column within the microfluidic substrate 250, described belowin connection with FIG. 17.

The interior side of the right casing section 200-1 includes a ridge 292of casing material that runs from the emitter end 204 and terminates atthe through-hole 212. When the casing sections 200-1, 200-2 are joined,the ridge 292 runs directly behind the groove 230 (FIG. 12) on theexterior side of the left casing section 200-2. The ridge 292 providesstructural support to the microfluidic cartridge 16. In addition, bybeing directly opposite the groove 230, the ridge 292 resists bending ofthe cartridge 16 by the guide pin 128 (FIG. 7) should a user prematurelyattempt to clamp the microfluidic cartridge 16 before the microfluidiccartridge 16 has fully reached the proper position. In addition, noportion of the microfluidic substrate 250 lies directly behind thegroove 230, as a precautionary measure to avoid having the guide pin 128bend the microfluidic substrate 250 in the event of a premature clampingattempt.

The interior side of the right casing section 200-1 provides the otherhalf of the gas well 276, the walls of which align with and abut thosedefining the well 276 on the left casing section 200-2. To enhance atight seal that constrains gas to within the gas well 276, a fastener orpin 296 (FIG. 14) tightens the connection between the casing sections atthe opening 298 adjacent the well 276.

FIG. 17 shows a left-side view of an embodiment of the microfluidicsubstrate 250. In brief overview, the microfluidic substrate 250 isgenerally rectangular, flat, thin (approx. 0.050″), and of multilayerconstruction. Formed within the layers of the microfluidic substrate 250is a serpentine channel 300 for transporting liquid. The microfluidicsubstrate 250 includes a trap region 302 and a column region 304. In theembodiment shown, the microfluidic substrate 250 has three microscopicfluidic apertures 306-1, 306-2, 306-3 (generally 306). One of thefluidic apertures 306-1 intersects the channel 300 at one end of thetrap region 302; another of the fluidic apertures 306-2 intersects thechannel 300 at the other end of the trap region 302. In this embodiment,the third fluidic aperture 306-3 is unused. Alternatively, the thirdfluidic aperture 306-3 can be used, for example, as a calibration port;an operator can run a calibration fluid. The channel 300 terminates atan egress end of the microfluidic substrate 250. The fluidic aperture306-2 at the “downstream” end of the trap region 302 is optionally usedas a fluidic outlet aperture, for example, during loading of the trapregion 302, and is optionally closed to fluid flow, for example, duringinjection of a loaded sample from the trap region 302 into the channel300.

The microfluidic substrate 250 also has a high-voltage input port 290(FIG. 16) and a pair of alignment openings 310-1, 310-2 (generally, 310)that each receives a peg that projects from an interior side of the leftcasing section 200-2. The alignment openings 310 help position themicrofluidic substrate 250 within the microfluidic cartridge 16. Thesize of the alignment openings 310, with respect to the size of thepegs, allows the microfluidic substrate 250 some play within themicrofluidic cartridge 16.

Some embodiments include multiple microfluidic substrates. For example,rather than a single microfluidic substrate 250, the microfluidiccartridge 16 can house a plurality of interconnected microfluidicsubstrates. Some example embodiments, having multiple substrates, aredescribed next, with reference to FIGS. 18A, 18B and 19.

FIG. 18A shows a portion of a device, according to one embodiment of theinvention, which has two (or more) fluidically connected microfluidicsubstrates 250-1, 250-2. The substrates 250-1, 250-2 include aseparations substrate (also referred to herein as a column tile orsubstrate) 250-2 and a trap tile (or load substrate) 250-1. Themicrofluidic substrates 250-1, 250-2 are coupled to each other viacouplers 322-1, 322-2, which are optionally aligned to ports in thesubstrates 250-1, 250-2 via fittings 325-1, 325-2, 326-1, 326-2(generally, fittings 325, 326.) The trap tile 250-1 has afluid-conducting channel 300-1 and the column tile 250-2 has a fluidconducting channel 300-2, which are connected via a coupler 322-2. Thelocations of the channels 300-1, 300-2 are illustrated, although one ofskill will understand that the channels 300-1, 300-2 are internal to thesubstrates 250-1, 250-2.

This example of a trap tile 250-1 has two or three fluidic apertures.Coupled about each fluidic aperture is a fitting 320-1, 320-2, 320-3(generally 320). The fitting 320-3 is optionally located at a dummyaperture; the dummy location may merely be used, for example,application of a clamping site. The fittings 320 serve to self-align thetips of the nozzles (e.g., nozzles 130 or 184 of FIG. 7 or FIG. 10,respectively) on the fluidics block 90 when the microfluidic cartridge16 is installed in the chamber, as described in more detail below. Thesefittings 310 can be made of metal, plastic, ceramic, or any combinationof these materials or other suitable materials. To couple the fittings320 to the substrate 250-1, they can be glued, fastened, fused, brazed,or a combination thereof, for example.

The tile 250-1 has a notch, or open spot 318, which, for example,provides access for a nozzle to directly contact an aperture of theseparations substrate 250-2. The aperture can be a dummy aperture, forexample, for substrates that do not require use of a fourth nozzlecontacting the substrate at the site of the dummy aperture. The notch318 optionally assists alignment, orientation and/or substrateidentifications, for example.

FIG. 18B shows a cross-section of the device of FIG. 18A, sliced throughthe trap tile 250-1 and column tile 250-2, two of the fittings 320-1 and320-2 and two couplers 322-1, 322-2 respectively associated with thefittings 320-1, 320-2. The channel 300-1 of the trap tile 250-1 extendsfrom the fitting 320-1 to the fitting 320-2. The couplers 322-1, 322-2(generally, 322) connect the trap tile 250-1 to the column tile 250-2.Each coupler 322 has an associated a pair of alignment fittings 325,326: one fitting 325-1, 325-2 on the underside of the trap tile 250-1,and the other fitting 326-1, 326-2 on the top side of the column tile250-2. The coupler 322-2 (as noted above) provides a channel (or lumen)324 for fluid passing through the channel 300-1 of the trap tile 250-1to reach the channel 300-2 of the column tile 250-2. The couplers 322can be made of metal, plastic, ceramic, or any combination of thesematerials and can be glued, fastened, fused, and brazed, or anycombination thereof, to the tiles 250-1, 250-2.

Preferably, however, in some embodiments, the couplers 322 are formed ofa deformable matter, and need not be permanently attached to eithersubstrate 250-1, 250-2; in some embodiments that have a swappable traptile 250-1, the couplers 326-1, 326-2 are fixedly attached to the columntile 250-2, to facilitate swapping of the trap tile 250-1. For example,a device optionally includes a cartridge having a housing with a slot, adoor, or other means to permit access to the cartridge for removableand/or insertion of substrate(s).

A deformable material is optionally any suitable material or materials,for example, a material similar to or the same as the material of thenozzles 130. Mechanical pressure alone is optionally used to provide afluid-tight seal between the trap tile 250-1, the couplers 322 and thecolumn tile 250-2, using, for example, a clamping device, such as theclamping device described below. Alignment-assisting features, such asthe fittings 320-1 and 320-2, are optionally included in the embodimentillustrated in FIG. 17 and in other embodiments, to align nozzles orfluidic connectors.

FIG. 19 is a view of an embodiment of a portion of an infusion devicehaving features similar to the multi-substrate device of FIG. 18A. Thedevice has two (or more) fluidically connected microfluidic substrates450-1, 450-2. The substrates 450-1, 450-2 include an injection substrate(also referred to herein as an infusion tile or substrate) 450-2 and asample tile (or sample-load substrate) 450-1. The microfluidicsubstrates 450-1, 450-2 are coupled to each other via couplers similarto the coupler 322 described above; the couplers are optionally alignedto ports in the substrates 450-1, 450-2 via fittings, such as thefittings 325, 326 described above. The sample tile 450-1 has afluid-conducting channel 400-1 onto which a sample is loaded, and theinfusion tile 450-2 has a fluid-conducting channel 400-2 through whichthe sample is injected into the inlet of a mass spectrometer.

The outer surface of the sample tile 450-1 optionally has one or morefittings 420-1, 420-2, 420-3, 420-4 (generally 420), similar to thefittings 320, to assist alignment of one or more nozzles deliveringand/or receiving fluids. In the illustrated embodiment, two ports of thesample tile 450-1 are optionally used to load a sample, and one port,for example, the port at fitting 420-3, is used to deliver a fluid toinfuse the sample, after the sample tile 450-1 is disposed in positionwith the infusion tile 450-2.

In some alternative embodiments, a secondary tile includes both aninfusion conduit and a trap column; the orientation of the tile isoptionally rotated to permit selection of the infusion conduit or theseparation column. In view of the description provided herein, one ofskill will recognize that various alternative embodiments can includevarying numbers of substrates each of which can include varying numbersand types of conduits. For example, a single substrate optionallyincludes two, or more, trap columns; for example, such a substrate canbe loaded with multiple samples and/or different trap columns can bedifferently configured, for example, with different packing materials.Multiple columns optionally reside in the same and/or different layersof a substrate, and are optionally oriented in parallel, perpendicular,or other directions relative to one another.

Infusion is used, for example, for delivery of a “neat” sample, withoutseparation, to a mass spectrometer. As known to one of skill, sampleinfusions are used, for example, to provide a relatively long analysisof a uniform sample. For example, a sample fraction can be infused formore than 30 minutes, allowing time for various MS experiments such asMS/MS, precursor ion or neutral loss scans and/or accurate massmeasurements, in positive and/or negative mode. Through fractioncollection and infusion, for example, a gain in data quality ispotentially obtained along with a time-saving benefit because theoriginal sample needs neither to be re-analyzed by re-injection norrequires pre-concentration. MS conditions are optionally more readilyoptimized for targeted ions, with different ion modes. Optionally,extended acquisition time permits summing of MS or MS/MS scans toimprove spectra.

Since infusion samples must generally be clean (e.g., no non-volatilesalt and limited amounts of volatile salt,) infusion can be difficultwhere only small sample volumes are available. Therefore, microfluidicdevices of the invention, which support nano-sample analyses, can beparticularly advantageous for infusion analyses.

In a multi-substrate device, according to some embodiments of theinvention, one or more of the substrates, such as the substrates 250-2,450-2 remain in a cartridge housing, while secondary substrates such asthe tiles 250-1, 450-1 are swapped for analyses of different samples.For example, samples may be loaded on several tiles, which are thenswapped in a cartridge for sequential analysis.

Multi-substrate devices have additional advantages. For example, thepacking of a column in a column tile and a column in a trap tile mayprogress more easily if the columns reside in different substratesrather than the same substrate.

Multi-substrate devices have other uses, in addition to those mentionedabove. For example, different substrates are optionally maintained atdifferent temperatures. For example, a temperature differential betweena trap column and a separation column may be more readily controlled ifthe columns reside in different substrates. In particular, this may bethe case where the substrates are formed of ceramic materials having arelatively high thermal conductivity. In some embodiments, activetemperature control is applied to one or more of the substrates. Forexample, one or more of the substrates can have heating and/or coolingfeatures, as described above.

FIG. 20, FIG. 21, and FIG. 22 illustrate the installation of amicrofluidic cartridge 16 in the clamping assembly 60. FIG. 20 shows across-section of the clamping assembly 60 with an empty chamber 120;FIG. 21 shows the cross-section after insertion of the microfluidiccartridge 16 into the chamber 120, but before clamping; and FIG. 22shows the cross-section after clamping. In each of FIG. 20, FIG. 21, andFIG. 22, the body 80 of the clamping assembly 60 houses a cam 350 and acam follower 352. The cam follower 352 is within a carrier 354; aportion of the cam follower 352 extends beyond the carrier 354 and abutsthe cam 350. One end of the plunger 126 is coupled to the cam follower354 within the carrier 354. A load spring 356 wraps around a rearwardsection of the plunger 126, and a return spring 358 wraps around aforward section of the plunger 126. Optionally, in some embodiments, acam is rotated to move a spring-loaded plunger into a closed position,and a secondary spring, such as the spring 358, provides load-balancingfor more consistent and accurate introduction of a desired force, suchas a force of 130 lbs.

In FIG. 20, the load spring 356 and return spring 358 are undamped; thelever 34 (FIG. 6), which is coupled to the cam 350, is in the open,unclamped position. In addition, springs 360-1, 360-2 (generally, 360)between the back wall 86 and the carriage 122 are likewise undamped.Projecting into the empty chamber 120, from the back wall of the endhousing 82, are a pogo pin electrical connector 144, gas nozzles 130-1,130-2 (a third nozzle being obscured), and the guide pin 128.

In FIG. 21, the microfluidic cartridge 16 is the chamber 120, with theemitter end of the cartridge entering the chamber 120 first. As themicrofluidic cartridge 16 enters the chamber 120, the guide pin 128slides along the groove 230 in the left casing section 200-2. Wheninsertion of the microfluidic cartridge 16 into the chamber 120 reachesits limit, the plunger 126 abuts the push block 210 on the right side ofthe microfluidic cartridge 16, and the guide pin 128 reaches thethrough-hole 212 (FIG. 12) at the end of the groove 230. If the guidepin 128 is not aligned with this opening, the microfluidic cartridge 16cannot be clamped. In one embodiment, the engagement of the arm 110(FIG. 5) with the nook (FIG. 11) in the upper edge of the microfluidiccartridge 16 determines how far the microfluidic cartridge 16 can enterthe chamber. In addition, the springs 124-1, 124-2 abut the right sideof the microfluidic cartridge 16. As in FIG. 20, in FIG. 21 the loadspring 356, the return spring 358, and the springs 360 are undampedbecause the lever 34 is in the open position.

In FIG. 22, the lever is closed, and the cam 350 causes the load spring356 and return spring 358 to compress and urge the plunger 126 againstthe push block 210. The spaced-apart bosses 260 (FIG. 13) on the otherside of the push block 210 distributes this force against themicrofluidic substrate 250. The force against the push block 210 movesthe carriage 122 and the microfluidic cartridge 16, together, towardsthe back wall 86 of the end housing 82. In addition, the spring 124-1operates to push the microfluidic cartridge 16 downwards and toward theback wall 86, while springs 360-1, 360-2 (FIG. 21) compress, resistingthe leftwards motion.

As a result of moving the carriage with the cartridge 16, the guide pin128 penetrates the through-hole 212 in the microfluidic cartridge 16.The nozzles 130 that project inward from the back wall 86 enter therespective nozzle openings 220 (FIG. 12) in the left casing section ofthe microfluidic cartridge 16, and press against the surface of themicrofluidic substrate 250. The urging force is sufficient to produce asealed fluidic pathway between the each nozzle and the fluidic aperture.The clamping also causes the pogo pins 144 to enter the window 224 onthe left casing section and make electrical connections with the arrayof electrical contacts 226.

In addition to establishing the fluidic interface between the nozzles ofthe fluidic block and the microfluidic substrate, and the electricalinterface between the pogo pins 144 and the array of electrical contacts226, this clamping action also establishes (1) the electrical interfacebetween the high-voltage pogo pin and the microfluidic substrate and (2)the fluidic interface between the gas nozzle and the microfluidiccartridge 16. FIG. 23 shows the high-voltage electrical cable 38 with apogo pin 380 entering the left casing section of the microfluidiccartridge 16, and a gas nozzle tip 382 entering the gas inlet port ofthe left casing section.

Some preferred embodiments of the invention entail apparatus of reducedcost and size relative to existing apparatus, such as existinganalytical equipment based on LC-MS. Miniaturization provides manypotential benefits in addition to size reduction, for example: improvingreliability; reducing the quantity and cost of reagents, and the cost ofused-reagent disposal; and improve performance reducing dispersion inLC-related components. While preferred embodiments, described herein,relate to liquid chromatography, one of skill will recognize that theinvention may be applied to other separation techniques.

While the invention has been shown and described with reference tospecific preferred embodiments, it should be understood by those skilledin the art that various changes in form and detail may be made thereinwithout departing from the scope of the invention as defined by thefollowing claims. For example, multiple substrates are optionallyattached to one another in a more or less permanent manner, for example,using glue or some other bonding mechanism.

1. An apparatus for chemical separations, comprising: a firstsubstantially rigid microfluidic substrate defining a first fluidicport; a second substantially rigid microfluidic substrate defining asecond fluidic port; and a coupler comprising a deformable materialdisposed between the first and second substrates, the coupler defining afluidic path in fluidic alignment with the ports of the first and secondsubstrates, wherein the deformable material is deformable relative to amaterial of the first substrate and a material of the second substrate.2. The apparatus of claim 1, wherein the first substrate further definesa separation column in fluidic communication with the first fluidicport.
 3. The apparatus of claim 1, wherein the second substrate furtherdefines a trap column in fluidic communication with the second fluidicport.
 4. The apparatus of claim 1, further comprising an alignmentfitting attached to the first substrate, to position the fluidic path ofthe coupler in fluidic alignment with the first fluidic port.
 5. Theapparatus of claim 1, wherein the coupler is fixedly attached to thefirst substrate.
 6. The apparatus of claim 1, further comprising ahousing that mechanically supports the first and second substrates. 7.The apparatus of claim 6, wherein the second substrate is removablydisposed in the housing, permitting exchange of the second substrate. 8.The apparatus of claim 6, wherein the housing supports the first andsecond substrates in a free-floating manner.
 9. The apparatus of claim1, further comprising a temperature control unit configured toindependently control a temperature of the first substrate and atemperature of the second substrate.
 10. The apparatus of claim 1,wherein an elastic modulus of the deformable material is substantiallylower than an elastic modulus of the first microfluidic substrate. 11.The apparatus of claim 10, wherein the first microfluidic substratecomprises sintered inorganic particles and the deformable materialcomprises a polymer.
 12. The apparatus of claim 11, wherein theinorganic particles comprise yttria-stabilized zirconium oxide.
 13. Amethod for performing chromatography, comprising: providing a firstsubstantially rigid microfluidic substrate defining a first fluidicport; providing a second substantially rigid microfluidic substrate,defining a trap column, and having inlet and outlet fluidic ports inrespective fluidic communication with an inlet and an outlet of the trapcolumn; providing a coupler defining a fluidic path and comprising amaterial deformable relative to a material of the first substrate and amaterial of the second substrate; loading a sample onto the trap column;disposing the loaded second substrate adjacent to the first substrate;disposing the coupler between the first and second substrates, influidic alignment with the outlet port of the second substrate and thefluidic port of the first substrate; urging the first and secondsubstrates towards each other to compress the coupler between the firstand second substrates and form a fluid-tight seal; and eluting thesample, via the coupler, from the trap column into the firstmicrofluidic substrate.
 14. The method of claim 13, further comprisinginfusing the eluting sample into a mass spectrometer upon exiting thefirst microfluidic substrate.
 15. The method of claim 13, furthercomprising separating the eluting sample in a separation column of thefirst microfluidic substrate.
 16. The method of claim 13, furthercomprising cooling or heating the second substrate relative to the firstsubstrate to provide a temperature differential between the first andsecond substrates while eluting the sample form the trap column.
 17. Amethod for fabricating a chromatographic apparatus, comprising:providing a first microfluidic substrate defining a separation column,and having inlet and outlet fluidic ports in respective fluidiccommunication with an inlet and an outlet of the separation column;providing a second microfluidic substrate defining a trap column, andhaving inlet and outlet fluidic ports in respective fluidiccommunication with an inlet and an outlet of the trap column; packingthe separation column with a first packing material; packing the trapcolumn with a second packing material different from the first packingmaterial; providing a coupler defining a fluidic path and comprising amaterial deformable relative to a material of the first substrate and amaterial of the second substrate; disposing the second substrateadjacent to the first substrate; disposing the coupler between the firstand second substrates, in fluidic alignment with the outlet fluidic portof the second substrate and the inlet fluidic port of the firstsubstrate; and providing a housing to mechanically support the first andsecond substrates.